Peach transcription factor PpERF.A16 gene, protein as well as recombinant expression vector and application thereof

A technology of transcription factors and expression vectors, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effects of reducing agricultural costs, increasing commodity value, and extending shelf life

Active Publication Date: 2018-09-28
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are relatively few studies on peach fruit ripening traits, only PG (polygalacturonase, polygalacturonase) gene involved in regulating peach fruit softening traits and ethylene upstream synthesis genes ACS and ACO involved in regulating peach fruit ripening traits has been initially confirmed
However, other structural genes and transcription factors consistent with peach fruit ripening phenotypes have not been reported so far.

Method used

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  • Peach transcription factor PpERF.A16 gene, protein as well as recombinant expression vector and application thereof
  • Peach transcription factor PpERF.A16 gene, protein as well as recombinant expression vector and application thereof
  • Peach transcription factor PpERF.A16 gene, protein as well as recombinant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of phylogenetic tree and related gene qRT-PCR analysis

[0044] 1. Download the ACS from the peach genome database (Rosaceae, http: / / www-RuxaA.Org) and other fruit trees (including apple, strawberry, papaya, orange and grape) (http: / / PosiZoM.jig.doe.gov) , ACO and ERF family members of the nucleotide and amino acid sequences. All genes were predicted in the National Center for Bioinformatics (NCBI; HTTPS: / / www.NcB.NLM.NIH.GOV / ), and the predicted genes were classified. The downloaded amino acid sequences were aligned by CulsTAL X, and a neighbor-joining (NJ)-based phylogenetic tree was constructed using the software MEGA 6 . the result shows:

[0045] 1.1 Six ACS gene family members were detected from the peach genome, which can be divided into three groups A, B, and C. Groups A and C consist of two subgroups ( figure 1 ). These genes were named PpACS.A1, PpACS.A2, PpACS.B1, PpACS.B2, PpACS.C1 and PpACS.C2 (Table 1).

[0046] 1.2 36 members o...

Embodiment 2

[0052] Example 2 PpERF.A16 Gene Isolation Cloning and Vector Construction

[0053] 1. Using the plant total RNA extraction kit of polyphenols and polysaccharides (purchased from TIANGEN Company, operated according to the kit instructions) to extract RNA from peach pulp, the first-strand cDNA obtained by reverse transcription was used to amplify Increase the full length of the PpERF.A16 gene. The primer pair for the amplification gene is: PpERF.A16-F1: (SEQ ID NO.3); PpERF.A16-R1: (SEQ ID NO.4). The 50 ul reaction system included 2 ul of the above primers, 17 ul of ddH2O, 25 ul of 2×Buffer, 1 ul of dNTP, 2 ul of cDNA template, and 1 ul of high-fidelity enzyme (Phanta Super-Fidelity DNA Polymerase) (purchased from Vazyme). The PCR reaction was completed on the Eppendor PCR amplification instrument according to the following program: 95°C, pre-denaturation for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 the...

Embodiment 3

[0055] Example 3 PpERF.A16 gene overexpression and construction of silencing vector

[0056] 1. According to the analysis of the multiple cloning site of the pCAMBIA-1301 vector and the restriction site on the coding region sequence of the PpERF.A16 gene, XbaI and HindIII were selected as endonucleases. According to the general principles of primer design, primers with restriction sites were designed with Primer 5.0 software, and the sequences of the primer pairs are as follows:

[0057] PpERF.A16-F1: ttggatccAGGAATGTGTGGCGGTGCTAT (SEQ ID NO. 3)

[0058] PpERF.A16-R1: aatctagaTTACGGAGCAGAAACGCGGTCG (SEQ ID NO. 4)

[0059] Using the extracted plasmid from the preserved bacterial liquid with correct sequencing as a template, clone the gene containing the restriction endonuclease site. The annealing temperature of PCR amplification is 60° C., and the PCR reaction system and amplification procedure are the same as in Example 1. The pCAMBIA-1301 vector was double-enzyme-digested...

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Abstract

The invention discloses a peach transcription factor PpERF.A16 and application thereof. The gene belongs to an ERF family member, and has a nucleotide sequence as shown in SEQ ID NO.1; the length of acoding sequence is 966bp, and 321 amino acids are coded; an amino acid sequence coded by the amino acids is as shown in SEQ ID NO.2. On the basis of genome and RNA SEQ analysis, an ethylene biosynthetic gene, 1-aminocyclopropane-1-carboxyl synthase and an AP2 / ERF transcription factor are separated and analyzed. Biological functional verification shows that the PpERF.A16 gene has a function of promoting ethylene synthesis. The finding of the PpERF.A16 gene provides new gene resources for promoting molecular breeding of the ethylene synthesis and provides new genetic resources for implementinggreen agriculture; development and utilization of the resources are favorable for improving the commercial value and the market competitiveness of peach fruits, prolonging the shelf life of fruits, reducing the agricultural cost and realizing environmental friendliness.

Description

technical field [0001] The invention relates to peach transcription factor PpERF.A16 gene, protein, its recombinant expression vector and application, and belongs to the field of plant genetic engineering. Background technique [0002] Fruit ripening refers to a complex and orderly process of a series of physiological and biochemical reactions that occur after fruit growth and development stop. Many aspects such as color, flavor, aroma, texture, etc. will change, and fruit ripening directly affects the commodity value of the fruit. Post-storage and market competitiveness. (Giovannoni, 2004; Li et al., 2010; Tian Shiping, 2013). According to whether there is a respiratory peak in the fruit during ripening, it is divided into respiratory climacteric and respiratory non-climactic type (Leli E Vre J, 1997). Tomatoes, apples, bananas, peaches, etc. have a sudden increase in respiration intensity and increased ethylene release during the fruit ripening process, all of which are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/11C07K14/415A01H5/00A01H6/74
CPCC07K14/415C12N15/8249
Inventor 张妤艳谷超郭志华俞明亮
Owner JIANGSU ACAD OF AGRI SCI
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